DNA damage is one of the major causes of the onset of aging in humans. Cells in our body are constantly exposed to DNA damage caused by external insults, such as exposure to radiation or chemicals, and by intrinsic stress generated by normal cellular activities such respiration, replication and progressive telomere erosion. Despite intensive study, the mechanisms by which the accumulation of DNA damage results in aging remain poorly understood. To investigate the effects of DNA damage in vivo we developed a mouse model in which DNA damage can be delivered exclusively to adult stem cells in an inducible manner. By depletion of critical telomere- associated proteins we can induce de-protection of chromosome ends in selected cell types. This results in uncapped chromosome ends, which are recognized as sites of DNA damage and initiate a DNA damage response. This response is indistinguishable from that observed in cells that have incurred DNA damage in other genomic regions. This proposal focuses on the mechanism of telomere dysfunction-induced aging, using as a model system the mouse intestine, colon and hair follicles, with the broad long-term objective of defining the critical mechanisms and pathways involved in the decline of regenerative potential observed in aging organisms. The experiments in Aim#1 will define the cell fate of stem cells upon the induction of telomere dysfunction. In this Aim we will test the hypothesis that adult stem cells are intrinsically resistant to DNA damage and that they accumulate mutations due to an active error prone DNA repair mechanism, the NHEJ pathway. The experiments in Aim #2 will investigate the physiological consequences of telomere dysfunction on tissue homeostasis. In this aim we take advantage of a lineage tracing approach that will allow us to define whether DNA damage results in the accumulation of damaged cells or alternatively, to the progressive depletion of critical progenitor cells. The experiments in Aim #3 will define the impact of telomere dysfunction in the context of checkpoint inhibition. This will allow us to define whether impaired tissue regeneration is the result of checkpoint activation (as from DNA damage) or from telomere dysfunction. Telomere erosion and progressive accumulation of DNA damage have been shown to play a significant role in the onset of human aging. Identifying the cellular and molecular mechanisms responsible for the decline in regenerative potential of tissues will provide crucial insight into the aging process.